Research Awardees: 2005
     Abstracts

Gene Therapy Collaboration:


David B. Levin, Ph.D.
Department of Biology, University of Victoria

Lay Progress Report (August 2006)
Rett syndrome, which is the second leading cause responsible for mental retardation in girls, has been determined to be caused by the change of a single but very important gene--Methyl-CpG binding protein 2 (MeCP2) gene. There is no current treatment approach available. Mutations in the mecp2 gene result in an MeCP2 protein that fails to regulate gene expression, resulting in "arrested development" of neuronal cells. A reasonable approach may be to deliver the normal gene back with the hope of rescuing the phenotype. We are part of an international collaborative research effort to develop a gene therapy strategy for Rett syndrome. Three research groups (Dr. David Levin and Dr. Kerry Delaney, at the University of Victoria, in Victoria, British Columbia; Dr. Jame Ellis and Dr. Peter Dirk, Hospital for Sick Children, University of Toronto, Toronto, Ontario; and Dr. Jude Samulski, Gene Therapy Centre, University of North Carolina, Chapel Hill, North Carolina) are working together to investigate parameters essential to the development of a viable gene therapy method. Our focus is on the use of virus-based vectors to deliver normal MeCP2 to neurons in the brains of mice with mutated mecp2, with the goal of restoring normal development of neuronal cells. Expected outcomes of our research collaboration include: i) delineation of the timing of neuro-development and architecture of neocortical cells in the brains of MeCP2-null mice; ii) characterization of the mechanisms that regulate the timing and tissue-specificity of mecp2 gene transcription; iii) identification of which isoform of MeCP2 (MeCP2E1 or MeCP2E2) works best as a therapeutic agent. Our collaboration with the other members of the team will also help determination of which vector system (Retrovirus, Lentivirus, Adeno-Associated Virus, and/or neural stem cells) offers the best potential for gene therapy delivery. Successful demonstration of the gene therapy in the MeCP2-null mouse model will provide data of fundamental importance for developing gene therapy for human RTT children.


James Ellis, Ph.D.
Hospital for Sick Children, University of Toronto

Lay Progress Report (August 2006)
Rett syndrome is caused by a mutation in the MeCP2 gene. This type of genetic disease may be treated by "gene therapy" that introduces the normal gene into affected cell types. Here, we propose to deliver the normal MeCP2 gene into brain cells using a specially designed retrovirus. This retrovirus vector will be tested in a mouse model of Rett syndrome, an objective that raises challenges that we have carefully identified and will resolve as follows. First, there are two different proteins encoded by the gene, called MeCP2E1 and MeCP2E2, and their relative importance is not yet well defined. To address this, we have generated retrovirus vectors encoding each protein separately. Second, MeCP2 must be carefully regulated because over-expression causes neurological disease. Therefore, we will test our existing ubiquitously expressed vectors in Neural Stem Cells (NSC) from MeCP2 null mice, and create novel MeCP2 minigenes regulated by their normal MeCP2 control elements. Third, gene therapy itself entails some risk that the virus may integrate into a chromosomal region that activates an oncogene and causes cancer. We have developed safety-enhanced lentivirus vectors that use "insulator" elements to prevent this insertional activation. Fourth, MeCP2 null mice are difficult to obtain in large numbers. To limit the number of mice required, we will culture "neurospheres" to produce expanded numbers of Neural Stem Cells (NSC), each of which generate many neurons when induced to differentiate. The NSC will be infected with MeCP2 retrovirus vectors, differentiated into neurons in culture, and the level of MeCP2 proteins tested. Fifth, delivery of the virus back into the brain can be performed in two different ways. NSC grown in culture can be manipulated outside the body ("ex vivo") and then the cells transplanted back into the brain where they will ultimately generate neurons with normal MeCP2. Alternatively, the virus can be delivered directly into a region of the brain where NSC normally reside ("in vivo"). We will compare both delivery methods to determine which is optimal for Rett syndrome gene therapy.

Our anticipated outcome is a proof of principle that gene therapy of NSC can produce neurons expressing correct levels of MeCP2. To this end, we have extensive experience in designing gene expression cassettes and gene therapy vectors for use in stem cells. Moreover, we already culture human and mouse NSC and deliver these cells into fetal mouse brains in utero using ultrasound-guided injection. We have convincing preliminary results demonstrating MeCP2 gene transfer, and proven expertise with all the procedures required for this therapeutically important study of gene therapy for Rett syndrome.


Jude Samulski, Ph.D.
Center for Gene Therapy, University of North Carolina

Lay Progress Report (August 2006)
One strategy for treating the Rett Syndrome may be to deliver a therapeutic gene to the brain of an affected individual. As a carrier for the therapeutic gene, adeno-associated virus (AAV) seems to be an excellent choice. This virus is unique in that it is not harmful to the human beings and has been tested in phase I clinical trials for several genetic disorders such as cystic fibrosis and hemophilia. At the moment, 8 different serotypes of AAV have been developed into gene carriers. In this study we aim to find a suitable serotype that can efficiently deliver genes to mouse brain. Genetic evidence supports that a MeCP2 gene mutation is a major cause of RTT. MeCP2 appears to maintain normal brain development by controlling its downstream targets. Based on this hypothesis, we have focused on reversing the abnormal control of the downstream targets by introducing the correct MeCP2 gene in an animal model. To achieve a safe and effective level of MeCP2 with AAV, we have focused on developing and optimizing a novel regulation system to help control MeCP2 gene expression. Over the past funding period, we have made significant progress as summarized below:

Identify an ideal AAV vector that specifically and efficiently transduces neurons globally. Using various serotype AAV vectors carrying a green fluorescent protein gene as a marker, we have generated preliminary data collaborating results obtained by others that serotypes 1 & 5 appear to transduce similar number of cells (avg. 3000), compared to AAV 2 (avg. 40) in the brain. The observation indicates that the current serotype vectors, while capable of mediating gene delivery, may not be optimal for efficient and global transduction of neurons and therefore for gene therapy of brain diseases. To develop more efficient carriers for gene delivery, we have taken a directed evolution approach which involves shuffling the available AAV serotypes to generate a library of AAV mutants. Our results support the notion that AAV mutants with remarkably enhanced delivery efficiencies for a particular target can be generated. We anticipate that such a powerful technology will allow us to create AAV mutants with highly improved efficiency of gene delivery to the brain.

Characterize a novel regulatable AAV expression vectors based on alternative splicing. We continue to use a novel regulation system based on alternative splicing to develop and optimize a marker AAV that specifically and efficiently transduces neurons globally.

Once steps 1 and 2 above have been completed, we will proceed with this system to deliver MeCP2 gene into mouse brain. We hope that the results obtained from these studies will start to develop a therapeutic approach for treating Rett syndrome.


John Aletta, Ph.D.
University of Buffalo
Regulatory Control of Cellular MeCP2 Function
$100,000
Research Sponsor: Ford Motor Company


Lay Progress Report (August 2006)
Experiments in the Aletta lab are aimed at understanding the cellular and molecular regulation of neuronal differentiation and development. Cell signals initiated by neurotrophins (NGF and BDNF) are involved in many aspects of neuronal development. We believe that one target of neurotrophin-mediated signals may be MeCP2. Neurotrophin activation of two kinds of regulatory enzymes known as kinases and methyltransferases is capable of producing chemical modifications, phosphorylation and methylation respectively, of MeCP2. We would like to know if, and how, these chemical changes of MeCP2 interact in living cells. By using easily manipulated cellular models of cell signaling and gene regulation to elucidate the biological consequences of neurotrophin-mediated changes in MeCP2 phosphorylation and methylation, our goal is to determine the normal cellular mechanisms that regulate MeCP2 function. It is hoped that this new information will be useful in the development of future pharmacological interventions for Rett Syndrome.


Ian Marc Bonapace, Ph.D.
University of Insubria (Italy)
The isolation of protein complexes involving MeCP2 and Np95: Understanding their roles in the structural organization of heterochromatin
$100,000
Research Sponsor: Pro Rett Ricerca


Lay Progress Report (August 2006)
Coming Soon


Uta Francke, M.D.
Stanford University
Is DLX5 a target of MeCP2 and, therefore, involved in Rett Syndrome?
$27,500
Research Sponsor: Eastern Development, LLC


Lay Progress Report (August 2006)
Coming Soon


Aristea Galanopoulou, M.D., Ph.D.
Albert Einstein College of Medicine
The role of the GABAergic system of the substantia nigra in the motor dysfunction of Rett Syndrome
$100,000


Lay Progress Report (August 2006)
Among the cardinal symptoms of Rett syndrome are the abnormal movements and motor function. The substantia nigra (SN) is one of the important nuclei involved in motor control. Neuropathologic studies on brains from patients with Rett syndrome demonstrate abnormalities in the SN. The differentiation of the SN is controlled by GABAA receptors and estrogens, the signaling pathways of which in certain cases interact. In this study we are testing, in Rett mice with mutated MECP2 gene, the hypothesis that there is an abnormal functional maturation of the GABAA receptors in the SN, which results in broader dysfunction of GABA and estradiol signaling during the early postnatal development of the SN, affecting calcium regulated gene expression. The information obtained from these studies will allow us to select drugs and interventions that may restore the normal pattern of maturation of the SN and hopefully improve the symptomatology in patients with Rett syndrome.


Peng Jin, Ph.D.
Emory University
Role of MeCP2 in small RNA-mediated gene regulation and identification of MeCP2-associated genomic regions using whole-genome BAC array
$100,000
Research Sponsor: Boston Wharf Company


Lay Progress Report (August 2006)
Rett Syndrome is a neurodevelopmental disorder mainly caused by mutations in the X-linked gene MECP2 and primarily affects females. MeCP2 is thought to selectively bind methyl-CpG-binding dinucleotides in mammalian genome and to block gene expression. Recent studies have shown that small RNAs (~20nt) play important role in transcriptional silencing, particularly in plant and yeast. In mammalian cells it has been shown that small RNAs could lead to DNA methylation and transcriptional repression. Mutations in MeCP2 affect its ability to block gene expression and may lead to aberrant patterns of gene expression in RTT. The predominant manifestation of central nervous system dysfunction in RTT suggests that MeCP2 plays critical roles in the development and stability of neurons. However, the genomic regions associated with MeCP2 remain to be defined and how the mutations in MeCP2 alter their association is still unclear. Small RNAs have been shown to play important roles in gene regulation. We are examining whether MeCP2 utilize small RNAs to repress transcription. In addition, we have tested NimbleGen CGH array and will use this array to identify the genomic target regions of


David Katz, Ph.D.
Case Western
Maturation of BDNF-dependent respiratory neurons in MeCP2 mutants
$88,000
Research Sponsor: CIBC World Markets Corporation

Lay Progress Report (August 2006)
Coming Soon


Janine LaSalle, Ph.D.
UC Davis
Investigation of novel MeCP2 target genes regulating neuronal maturation
$100,000
Research Sponsor: American Commercial Claims Administrators, Inc.

Lay Progress Report (August 2006)
Rett syndrome is cause by mutations in the gene MECP2 that encodes a protein, methyl-CpG protein 2 (MeCP2). Elevated MeCP2 expression is acquired in individual neurons within the brain beginning in infancy and progressing throughout childhood. The function of MeCP2 in the developing brain is unclear at this stage, but the mutations in Rett syndrome and the Mecp2 deficient mouse model provide evidence that MeCP2 is essential for mature neuronal function.

MeCP2 is predicted to be a regulator of other genes in maturing neurons, but finding these genes is complicated by the complexity of cells and genes in the brain. In a previous study funded by the RSRF, a group of genes, called "ID" for "Inhibitors of Differentiation" were found to show altered expression when MeCP2 activity was blocked in neuronal cultures. ID genes are well-characterized regulators of cell differentiation (maturation) and the increased expression of these genes could help explain the why neurons appear to be immature in Rett syndrome brains.

Our recent publication (Peddada et al., Human Molecular Genetics, 2006) has demonstrated significantly increased levels of all four IDs (ID1, ID2, ID3, ID4) in Rett syndrome brain and Mecp2 deficient mouse brain. To further test the hypothesis that IDs are important in the pathogenesis of Rett syndrome, we are breeding Mecp2 deficient mice to Id1 and Id3 deficient mice to determine if reducing the dosage of ID genes may lessen the severity of disease.


Jeffrey Macklis, M.D., D.H.S.T
Recent research has revealed that a defect in a gene called MECP2, which encodes a protein that suppresses expression of other genes ("transcriptional repressor"), causes Rett syndrome. The discovery of MECP2 mutations as the overwhelming cause of Rett syndrome enabled a new era of cellular and molecular analysis, and understanding of the mechanisms of Rett syndrome. An important next research goal will be to find the specific target genes that MeCP2 regulates in individual affected nerve cells, because MeCP2 normally regulates its target genes during development and function of the nervous system, and abnormal expression of target genes directly or indirectly causes Rett syndrome.

Our previous work demonstrates that MeCP2 is involved in the maintenance and maturation of brain neurons, including their connections, and the stabilization of neurons with long axons, rather than the early development or movement of neurons as the brain is initially formed. These previous results show that "pyramidal neurons" in cerebral cortex layer 2/3 in Mecp2 mutant mice are smaller and their dendrites are less complex than those in normal mice. In addition, our recent work using genetic and physical chimeric mice (mixing one type of neuron with another type of brain), shows that a normal environment does not eliminate the abnormalities of transplanted Mecp2 mutant neurons, indicating that the lack of MeCP2 in neurons themselves (rather than in surrounding cells lacking MeCP2) is the central reason for their abnormalities.

Based on our previous work, we have pursued two complementary approaches for the identification and molecular analysis of target genes of MeCP2 in these important cortical neurons (callosal projection neurons; CPN), using both microarray and chromatin immunoprecipitation (ChIP) approaches. Using 'microarrays', we can investigate expression levels of many genes in cells at once. By comparing gene expression patterns in normal neurons with those in neurons from Mecp2 mutant mice, we found that 18 genes were significantly increased and 21 genes were decreased in Mecp2-mutant ("null", lacking Mecp2 function) CPN. One of them is increased 6.2-fold in Mecp2-null CPN compared to wild-type CPN. We tentatively named this gene MeCP2 Target Gene 1 (Mtg1). To confirm this result, we performed RT-PCR, another method to investigate gene expression. Importantly, we could detect the gene expression difference only when we used pure CPN, but not when we used the whole cortex, demonstrating that the use of pure cell populations can detect new direct or indirect MeCP2 target genes hidden by cellular heterogeneity.

We have also pursued the identification of MeCP2 target genes using a pure cell population of CPN by a ChIP-based approach. ChIP is an approach to determine the location on chromosomes where molecules like MeCP2 bind, and thus, which genes they regulate. So far, we have found approximately 200 MeCP2 binding DNA fragments. Using the mouse genome database, we mapped those DNA fragments onto the genome. The MeCP2 binding sequences we obtained are distributed in special areas on each of the mouse chromosomes. Approximately 14% of these DNA fragments were found to be within 20 kb "upstream" from a transcriptional start, suggesting that MeCP2 may bind these regions and control the expression of these genes in normal CPN.

We are now confirming these results from the microarray experiments using other methods, such as RT-PCR, and ChIP analysis.


David Paterson, Ph.D.
Children's Hospital, Boston
The medullary serotonergic system and respiratory dysfunction in Rett Syndrome
$25,000 (Pilot Project)

Lay Progress Report (August 2006)
Coming Soon


Thomas Seyfried, Ph.D.
Boston College
Gene-environmental interactions in the metabolic control of Rett Syndrome in Mecp2 mice using a ketogenic diet
$77,000
Research Sponsor: Connors Family Charitable Gift Fund

Lay Progress Report (August 2006)
We conducted the first evaluation of the ketogenic diet on behavioral, metabolic, and neurochemical parameters in adult Mecp2 308/Y Rett mice and in their normal male littermates. The ketogenic diet we administered was KetoCal®, a nutritionally balanced soybean oil-based diet for managing seizures in children with epilepsy. The ketogenic diet is effective in managing seizures and improving behavior in some children with Rett syndrome. KetoCal® was administered to the mice in restricted amounts as recommended for use in children with epilepsy. The normal and the Rett mice were separated into two groups: 1) a high carbohydrate, low fat standard chow diet group fed unrestricted (SD-UR), and 2) the KetoCal® diet group restricted to achieve a 25% body weight reduction (KC-R). The number of normal and Rett mice in each dietary group was 7 and 4, respectively. We measured; 1) food intake and body weight as indicators of metabolic state, 2) rotorod performance, anxiety (exploration of novel environment), proprioception, and behavioral seizures as indicators of motor coordination and behavior, and 3) cerebellar lipid composition as an indicator of neural structure and cellular integrity. We found that body weight in the SD-UR groups was significantly higher (P < 0.01) in Rett mice (40.4 + 2.2 g) than in normal mice (34.4 + 0.8 g), despite similarities in food intake. These findings suggest that the basal metabolic rate is abnormal in the Rett mice. The Rett mice showed no behavioral seizures, proprioceptive defects, or neurodevelopmental abnormalities when compared to the normal mice. However, rotorod performance (time on bar) in the SD-UR groups was significantly less in the Rett mice (6.2 + 1.8 sec) than in the normal mice (32.9 + 9.0 sec) (P < 0.01). Although the restricted KetoCal® diet significantly improved rotorod performance in the normal mice (58.1 + 1.9 sec), KetoCal® had no significant effect on rotorod performance in the Rett mice (5.9 + 2.4 sec). KetoCal® reduced anxiety in the normal mice, but did not reduce anxiety in the Rett mice. No differences were found between the normal and Rett mice for the content or distribution of cerebellar gangliosides, neutral lipids (cholesterol, phosphatidylethanolamine, phosphatidylcholine, cerebrosides, and ceramides), and acidic lipids (cardiolipin, phosphatidylserine, phosphatidylinositol, and sulfatides). We conclude that KetoCal® did not improve motor coordination or reduce anxiety in the Rett mice relative to normal mice, and that the motor and behavioral abnormalities in the Rett mice were not associated with noticeable changes in the content or distribution of cerebellar lipids. Studies are in progress to determine if the abnormality in basal metabolic rate in Rett mice is related to abnormalities in mitochondria.


Yi Eve Sun, Ph.D.
UCLA
MeCP2 glycosylation and phosphorylation, the Yin and Yang aspects of MeCP2 post-translational modifications
$100,000
Research Sponsor: Robert and Adele Schiff Foundation

Lay Progress Report (August 2006)
Coming Soon


RSRF Post-Doctoral Fellowship Awards


Feixia Chu, Ph.D.
UCSF
Mentor: Barbara Panning and A.L. Burlingame
Unraveling aberrant epigenetic gene regulation in Rett Syndrome using mass spectrometry
$100,000

Lay Progress Report (August 2006)
RTT has recently been attributed to mutations in a gene encoding the methyl CpG binding protein 2 (MeCP2). MeCP2 interacts specifically with methylated DNA CpG islands via its methyl-binding domain (MBD), and its transcriptional repression domain (TRD) recruits a co-repressor complex, including Sin3A and histone deacetylases. Deacetylation of core histones results in chromatin compaction and transcriptional repression of the target genes. Furthermore, transcriptional repression in vivo could be relieved by deacetylase inhibitor trichostatin A (TCA), underlining the MeCP2-orchestrated chromatin structure alteration as an essential component in this gene expression regulation process. I have set off to use mass spectrometry based proteomic approach to identify changes in posttranslational histone modifications and in global protein expression patterns in MeCP2 knock-out mouse brain. A direct comparison of mass spectrometric analysis on histones at the peptide level has revealed a substantial discrepancy in histone posttranslational modifications between wt and ko mouse, including an up-regulation on H4 Lys20 trimethylation, and a down-regulation on acetylation of histone H4 Lys31 and phosphorylation of H4 Ser47. More comprehensive data processing and analysis is currently underway.


Gregory Pelka, Ph.D.
Children's Medical Research Institute, Australia
Mentor: John Christodoulou
Investigation of the impact of regionalized Mecp2 deficiency and the manifestation of the RTT phenotype using chimera analysis
$100,000

Lay Progress Report (August 2006)
Coming Soon


Georg Stettner, M.D.
Georg August University, Germany
Mentor: Peter Huppke
Analysis of breathing abnormalities in Rett patients and MeCP2 knockout mice and development of therapeutic strategies
$100,000

Lay Progress Report (August 2006)
Most girls suffering from Rett syndrome (RTT) develop severe breathing abnormalities, which have a significant influence on the life quality of the affected girls and also their families. Furthermore, breathing arrhythmias are seen as a main cause of sudden and unexpected death in RTT. Since only little is known about the mechanisms leading to these breathing abnormalities, the aim of our research project is to understand the causes of this breathing disorder to have a starting point for the development of effective future therapies.

During the first year of RSRF funded research we investigated the breathing abnormalities in a RTT mouse model, the so-called Mecp2-/y knockout mouse. We used a working heart-brainstem preparation (WHBP) that represents the entire neuronal breathing network located in the mice brainstem. We found that brainstem preparations of RTT mice display the breathing abnormalities seen in living RTT mice and moreover, that these abnormalities are strikingly similar to those seen in RTT patients. We could further demonstrate that in RTT mice the early expiratory phase, also called postinspiratory phase, was longer and in its duration highly variable when compared to control mice. The cause for these findings might be a defect in control mechanisms located in neurons (nerve cells) of the brainstem. Further experiments are necessary to proof this hypothesis. These results are of high clinical interest since most of the respiratory abnormalities in RTT patients like repetitive apneas, breath holding spells, Valsalva's maneuvers, and loss of speech are potentially linked to a defect in the brainstem respiratory network.

We also investigate the breathing pattern of RTT girls from different age groups and with different MECP2 mutations by using a mobile monitor. This allows us to perform the measurements at home in the patients' usual environment avoiding hospitalization. So far, 12 girls with RTT have been studied. We will go on and monitor at least 25 patients. These data will illustrate the typical breathing pattern of RTT patients in their daily environment and together with results from the RTT mice breathing data will be the basis for designing a clinical trial to treat breathing abnormalities in RTT girls.